Introduction

Nanotechnology enables the creation of novel nanostructures with at a minimum one measurement < 100 nm.1 Because of their high surface area to volume ratio, nanostructures display distinctive physical and chemical characteristics when compared to their normal-sized counterparts.2 Nanoparticles have shown the possibility to be harnessed in numerous fields, including agriculture, medicine, photocatalytic dye degradation, the food industry, cosmetics, paints, and energy.3,4 The NiO nanoparticles are being exploited in several areas, for instances battery manufacture, light-emitting diodes, electrochemical sensors, biosensors, automobile rear-view mirrors, color enamels, and nanowires.5,6 NiO nanoparticles also effectively inhibited a broad range of fungal and bacterial pathogens.7,8. Nonetheless, several findings have revealed that due to unique features, NiO nanoparticles may also pose a significant risk to human and environmental health.9,10,11.

Graphene is a honeycomb-like carbon material that has recently appeared as one of the most prominent two-dimensional (2D) structures.12 Graphene derivatives, such as graphene oxide (GO) and reduced graphene oxide (rGO), possess exceptional electrical, thermal, mechanical, optical, and biological properties.13,14,15 Due to these remarkable characteristics, GO and rGO are appropriate structures for their integration with metal oxide nanoparticles (e.g. ZnO, CuO, NiO, TiO2, etc.,) to create unique nanohybrids of superior properties when compared to their single components.16,17,18.

Scientists are perpetually trying to augment the biological application of NiO nanoparticles by tuning their physicochemical properties through different methods, such as doping and integration with graphene derivatives.19,20 For instance, NiO decorated on rGO sheets (NiO@rGO) nanohybrids have shown improved performance in lithium-ion batteries, supercapacitors, environmental remediation, and nanomedicine.18,19,20,21 However, the growing applications and production of NiO@rGO nanohybrids may also pose a risk to human and environmental health, as nanohybrids can freely enter the cells and interact with biomolecules.21,22 Hence, studies on the consequences of such nanocomposites on human health need to be investigated before their appropriateness for copious applications, including bioimaging, biosensing, drug delivery, and the treatment of cancer.23,24 For example, a recent study reported that GO-Au nanohybrids accumulated in Daphnia magna and caused internal damage to the gut.25 Another study discovered that ZnO/TiO2 conjugated carbon-based nanohybrids generate adverse effects in coastal marine algae (Thalassiosira pseudonana) through the oxidative stress pathway.3.

The majority of research mainly focuses on the toxic effects of single types of nanoparticles, indicating an important knowledge gap in exploring the biological interaction of nanohybrids or nanocomposites. There is plenty of research on the biological effects of NiO or graphene derivatives alone. However, there is a scarcity of research on the consequences of NiO@rGO nanohybrids on biological systems. We designed this research to examine the toxic outcomes of synthesized NiO@rGO nanohybrids in normal rat kidney cells (NRK-52E). The NiO@rGO nanohybrids were produced by a simple hydrothermal route. Characterization of prepared samples was carried out by XRD, TEM, SEM, EDS, and DLS techniques. MTT, trypan blue, and cell morphology assays were applied to assess the cytotoxicity of NiO@rGO nanohybrids in NRK-52E cells. The assessment of the oxidative stress response of NiO@rGO nanohybrids involved measuring the level of glutathione (GSH) and reactive oxygen species (ROS) in NRK-52E cells. The apoptosis potential of NiO@rGO nanohybrids against NRK-52E cells was studied by measuring the chromosomal condensation, caspase-3 enzyme, cell cycle phases, and acridine orange/ethidium bromide (AO-EtBr) dual probing. To evade cell-type-specific responses, the effects of NiO@rGO nanohybrids were also examined in human lung cancer (A549) cells and human umbilical vein endothelial cells (HUVECs) through the exploration of cell viability and ROS levels. These cell lines (NRK-52E, A549, and HUVECs) are established in vitro models for toxicity assessment of nanostructured materials and for providing their detailed mechanistic pathways.26,27,28,29.

Experimentation

Synthesis of NiO@rGO nanohybrids

The NiO@rGO nanohybrids were prepared through a simple hydrothermal method adopted from Vivek et al.20 with some specific modifications. Briefly, 0.1 mM of Ni(NO3)2 was homogeneously mixed with ethylene glycol (40 mL) and deionized water (40 mL) to get solution (A) In another container, 10 mg of GO was suspended in 100 ml of deionized water and sonicated for 2 h to get solution (B) Then, solution B was mixed with solution A and transferred to a Teflon-coated autoclave and incubated for 12 h at 170 °C. The autoclave was then allowed to cool down to room temperature. Finally, black precipitate was collected by centrifugation. The obtained nanocomposite was desiccated at 60 °C for 24 h.

Characterization of NiO@rGO nanohybrids

Phases and crystal structure of NiO@rGO nanohybrids were analysed using X-ray diffraction (XRD, PANalytical X`Pert Pro) (Malvern Instruments, UK). The structural characterization of prepared samples was further carried out by field emission transmission electron microscopy (FETEM, JEM-2100, JEOL, Inc., Tokyo, Japan) and field emission scanning electron microscopy (FESEM, JSM-7600 F, JEOL, Inc.). The energy dispersive X-ray spectroscopy (EDS) was applied to estimate the elemental composition and mapping of prepared nanohybrids. The dynamic light scattering (DLS, Zeta-Sizer Nano-HT) (Malvern Instruments, UK) was applied to characterize the agglomeration and surface charge of nanohybrids in cell culture medium.

Cell culture and cell exposure

The NRK-52E, HUVECs, and A549 cells were obtained from ATCC (Virginia, USA). Cell lines were cultivated in DMEM containing 10% fetal bovine serum (FBS), 13.5 g/L sodium bicarbonate, and an antibiotic-antimycotic solution (50 units/mL penicillin, 50 µg/mL streptomycin, and 0.25 µg/mL Amphotericin-B). The cells were incubated in a humidified incubator with 5% CO2 at 37 °C. After achieving about 70–80% confluence, cells were collected using trypsin-EDTA and plated in different culture plates to perform the nanotoxicity experiments.

The NiO@rGO nanohybrids were suspended in DMEM to form a stock solution of 1 mg/mL. The initially prepared suspension of NiO@rGO nanohybrids was then diluted to the required dosages (1–200 µg/mL) and exposed to the cells for different time intervals (24–72 h). To avoid agglomeration prior to cell exposure, nanohybrid solutions were sonicated in a water bath sonicator for 15 min. Control cells were used in each experiment as a comparison to NiO@rGO nanohybrid-treated cells.

Biochemical assays

The MTT assay was applied to examine the cell viability against NiO@rGO nanohybrids following the protocol of Mossman with some specific changes.30,31 Cytotoxicity of NiO@rGO nanohybrids was examined through typan blue dye exclusion assay.30,32 Morphology of the control and NiO@rGO nanohybrid-treated cells was assessed by an inverted phase-contrast microscope (Olympus) at a 20X magnification. The ROS level was assayed using a 2,7-dichlorofluorescin diacetate (DCFDA) dye.33,34. Cell extract was prepared following the method outlined in our earlier work.35 The Ellman`s protocol was applied to estimate the GSH content.36 The caspase-3 enzyme activity was assayed using a colorimetric kit (BioVision, USA). Chromosomal condensation following exposure to NiO@rGO nanohybrids was examined using the 4’, 6-diamidino-2-phenylindole (DAPI) probe.30 The apoptosis response of NiO@rGO nanohybrids in the NRK-52E cells was assessed through acridine orange/ethidium bromide (AO/EtBr) dual probing.38 Cell cycle analysis was done at a flow cytometer (FACS CantoTM II, BD Biosciences, USA) using a propidium iodide (PI) fluorescent probe.37 Protein content was measured applying bicinchoninic acid (BCA) assay.39 A concise explanation of each biochemical parameter is presented in supplementary information.

Statistical analysis

All the quantitative data were presented as the mean ± SD of three individual tests. Results were assessed through one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. The p-value less than 0.05 was assigned as statistically significant.

Results and discussion

XRD study

The XRD pattern of NiO@rGO nanohybrids revealed diffraction peaks at 2θ values of 37.28, 43.37, 62.81, 75.32, and 79.38, credited to the indication of (111), (200), (220), (311), & (222) planes, respectively (Fig. 1). The XRD pattern of prepared nanohybrids is indexed to the face-centred cubic structure of NiO (JCPDS No. 65-2901).40 The sharp diffraction peaks show a high degree of crystallinity in the synthesized nanohybrids. The Scherrer formula was implemented to estimate the size of nanohybrids.40 The mean particle size of NiO@rGO nanohybrids was about 28 nm. The rGO peaks were not visible in NiO@rGO nanohybrids, indicating the prevention of restacking of rGO layers due to the anchoring of NiO nanoparticles on rGO sheets.41 The XRD result of this work was consistent with earlier reports.42.

Fig. 1
figure 1

XRD spectra of NiO@rGO nanohybrids.

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TEM and SEM characterizations

The microstructure of NiO@rGO nanohybrids was further investigated using FETEM and FESEM. Figure 2A depicted that spherical NiO nanoparticles were tightly anchored on thin layers of RGO. The mean particle size of prepared nanocomposites calculated from the TEM micrograph was almost 24 nm. The high-resolution micrograph of TEM indicates crystal phases of synthesized NiO@rGO nanohybrids (Fig. 2B). Figure 2C represents the elemental analysis of NiO@rGO nanohybrids. Figures 3A–C show the SEM micrographs of NiO@rGO nanohybrids at different magnification scales. Similar to TEM images, SEM micrographs also depicted that NiO nanoparticles have been firmly appended to the rGO sheets. The median size of NiO@rGO nanohybrids measured from SEM was about 25 nm. The composition of NiO@rGO nanohybrids was measured by EDS (Fig. 3D). These spectra depicted a nickel (Ni) peak at 0.9 and 8 keV, an oxygen (O) peak at 0.5 keV, and a carbon (C) peak at 0.3 keV. These findings are aligned with previous work.43 Figures 4A–D illustrate the elemental mapping of NiO@rGO nanohybrids. These micrographs also suggested the existence of nickel, carbon, and oxygen in prepared nanohybrids without impurities.

Fig. 2
figure 2

NiO@rGO nanohybrids TEM characterization (A) low-resolution, (B) high-resolution TEM micrographs, and (C) EDS analysis.

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Fig. 3
figure 3

SEM analysis of NiO@rGO nanohybrids. (A–C) SEM micrographs of NiO@rGO nanohybrids at different magnifications and (D) elemental composition of NiO@rGO nanohybrids by EDS.

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Fig. 4
figure 4

NiO@rGO nanohybrids elemental mapping by SEM. (A) An electron micrograph, (B) nickel, (C) carbon, and (D) oxygen mapping.

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DLS study

Characterization of nanoscale materials in physiological media such as cell culture medium is crucial before their toxicity or biomedical experiments.44 In this work, the aqueous behavior of NiO@rGO nanohybrids was explored in cell culture medium (DMEM) using DLS. The median hydrodynamic size of NiO@rGO nanohybrids in DMEM was 80.84 ± 13.16 nm (Fig. 5A), which is about 3–4 times higher than particle sizes obtained from XRD, TEM, and SEM, suggesting the agglomeration of nanohybrids in an aqueous state. This is due to the fact that DLS computes Brownian motion, and the subsequent size dissemination of a group of nanoparticles in suspension results in a mean hydrodynamic diameter that is typically larger than the TEM diameter, as it represents a dried layer of nanoparticles on the TEM grid. In DLS estimation, nanoparticles tend to agglomerate in an aqueous state, leading to the size of gathered particles rather than single ones. A higher hydrodynamic size of nanoscale structures compared to the size calculated from powder form has also been observed in earlier studies.45,46 The zeta potential of NiO@rGO nanohybrids in DMEM was found to be around − 17.00 ± 3.81 mV, indicating that prepared nanohybrids are fairly stable in cell culture medium (Fig. 5B).

Fig. 5
figure 5

Aqueous characterization of NiO@rGO nanohybrids in DMEM by DLS. (A) hydrodynamic size and (B) surface zeta potential.

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NiO@rGO nanohybrids generate cytotoxicity in NRK-52E cells

The MTT assay, trypan blue assay, and cell morphology study are the most common in vitro approaches for determining the cytotoxicity of drugs, chemicals, or pollutants.47,48 These assays are also crucial in determining the toxicity or biocompatibility of unique nanostructured materials prior to their animal testing.49 It is also feasible to research the plausible mechanisms of nanoparticle-induced toxic effects through these in vitro studies.50 In this study, cytotoxicity of NiO@rGO nanohybrids was determined in NRK-52E cells through these assays. NiO@rGO nanohybrids were exposed to NRK-52E cells at various doses (1–200 µg/mL) for 24–72 h. MTT outcomes demonstrated that NiO@rGO nanohybrids exert cytotoxic effects on NRK-52E cells in a dose- and time-reliant mode (Fig. 6A). The IC50 values of NiO@rGO nanohybrids in NRK-52E cells were 186.24, 23.79, and 9.39 µg/mL after 24, 48, and 72 h, respectively (Table 1). Cytotoxicity of NiO@rGO nanohybrids in NRK-52E cells was also assessed through a trypan blue test. Figure 6B showed that NiO@rGO nanohybrids induce cytotoxic effects in NRK-52E cells, and the intensity of cytotoxicity was intensified with increasing the dosage and exposure time of nanohybrids. The IC50 values of NiO@rGO nanohybrids for the trypan blue assay in NRK-52E cells were 180.43, 24.09, and 12.99 µg/mL for 24, 48, and 72 h, respectively. Cytotoxicity data from the trypan blue dye exclusion experiment supported the findings from the MTT assay, demonstrating that both approaches generated almost similar results when examining the cytotoxic effects of NiO@rGO nanohybrids in NRK-52E. Morphology of NRK-52E cells was further scrutinized following exposure to various dosages (25–100 µg/ml) of NiO@rGO nanohybrids for 24, 48, and 72 h. Morphology of NRK-52E cells against NiO@rGO nanohybrids exposure is presented in Figure 6C. The NiO@rGO nanohybrid-treated group showed significant cell death characterized by rounded floating cells with low cell density. Previous studies indicated that NiO NPs triggered cell death in a range of human cells, such as breast, liver, lung, kidney, and colon cells.51,52,53,54 Some recent work also reported the cytotoxicity of NiO- or rGO-based nanocomposites. For instance, Rajivgandhi et al. found that graphene-NiO nanocomposites generate cytotoxicity in human prostate (LNCaP) and human lung cancer (A549) cells.55 Periyasamy et al. reported that Ag nanoplate-decorated rGO (Ag-rGO) nanocomposites exert cytotoxic effects in SiHa cervical cells.56 Moreover, Krishnan and co-workers showed that ZnFe2O4/rGO nanocomposites were cytotoxic for A549 cells.57 As with most published research in the field of nanostructure toxicology, the high concentration-generated cytotoxicity reported in this work may be challenging to translate to a reasonable human exposure setting. However, we believe these data are poised to provide a motivation for other researchers to invent mechanistic pathways engaged in NiO@rGO nanohybrids that generate apoptosis in cell culture model systems and offer information for this essential scarcity in this swiftly budding area of human exposure concern.

Fig. 6
figure 6

Dose- and time-dependent cytotoxicity of NiO@rGO nanohybrids in NRK-52E cells. (A) MTT, (B) trypan blue, and (C) cell morphology assays of treated and control cells. *p < 0.05 vs. control.

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Table 1 IC50 values of NiO@rGO nanohybrids at different time-intervals in NRK-52E cells.
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NiO@rGO nanohybrids induced oxidative stress in NRK-52E cells

Intracellular ROS production and the resultant oxidative stress are primary causes of nanotoxicity. Engineered nanostructures, due to their tiny size, high specific volume-to-area ratio, and strong surface reactivity, augment the ROS generation in the cells, triggering irreparable injury through the oxidation of DNA, RNA, and proteins.58,59,60 Here, we researched the job of NiO@rGO nanohybrids in ROS elevation in NRK-52E cells. Fluorescent microscopic images showed that NiO@rGO nanohybrids treated cells display a brighter green fluorescence of DCF dye (an indicator of ROS generation) dose-dependently than control cells (Fig. 7A). In the same way, quantitative data showed that NiO@rGO nanohybrids significantly increase the ROS levels in NRK-52E cells as compared to controls. In comparison to the 100% DCF fluorescence in control cells, the cells that were treated with 1, 5, and 25 µg/ml of NiO@rGO nanohybrids had 130%, 272%, and 367% more fluorescent intensity, respectively (Fig. 7B). Earlier work revealed that NiO nanoparticles elevate ROS levels in A549 and HepG2 cell models.53,61 There are also some studies showing that metal oxide-rGO nanohybrids, such as Bi2O3-rGO, and CuO-rGO nanohybrids, generate ROS in biological systems.30,62.

Fig. 7
figure 7

Oxidative stress response of NiO@rGO nanohybrids in NRK-52E cells. (A) Fluorescent microscopic images of DCFH-DA probed control and treated cells. (B) Quantitative analysis of fluorescence intensity of DCF probe (an indicator of intracellular ROS level) in control and treated cells. (C) Quantitative analysis of GSH content in control and treated cells. *p < 0.05 vs. control.

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In cells, glutathione (GSH) acts as a primary antioxidant resistance molecule against ROS-induced cell damage.63 A depletion in intracellular GSH content is also associated with an early event of apoptosis.64 In this study, the effect of NiO@rGO nanohybrids on the GSH content of NRK-52E cells was further explored. NRK-52E cells were treated with nanohybrids at 1, 5, and 25 µg/mL doses for 48 h. The outcomes represented that increasing the concentration of NiO@rGO nanohybrids reduced the amount of GSH (Fig. 7C). These results indicated that NiO@rGO nanohybrids cause cytotoxicity in cells through the oxidative stress pathway. A further comprehensive analysis of oxidative stress biomarkers at the molecular level might provide a better rationale for the present findings. The exploration of oxidative stress biomarkers through molecular analysis may also enhance our understanding of cellular damage and its implications for various diseases.

NiO@rGO nanohybrids induced chromosomal condensation and caspase-3 enzyme activation in NRK-52E cells

Apoptosis is often characterized by chromatin condensation, remodeling, and nuclear fragmentation.65 In this work, the effect of NiO@rGO nanohybrids on chromosomal condensation was examined by DAPI staining using fluorescent microscopy. Results showed that control cells had intact nuclear chromatin, whereas treated cells had condensed nuclear chromatin and fragmented nuclei (Fig. 8A). The subsequent phase in the apoptotic process involves caspase-3 activation.66 The function of caspases is to regulate the process of apotosis.67 The potential apoptosis response of NiO@rGO nanohybrids in NRK-52E cells was further assessed by assaying caspase-3 enzyme performance. The NRK-52E cells were dosed to various levels (1, 5, 25 µg/mL) of NiO@rGO nanohybrids for 48 h. As we can see in Figure 8B, NiO@rGO nanohybrids induced caspase-3 enzyme activity concentration-dependently.

Fig. 8
figure 8

(A) Chromosomal condensation (DAPI staining) & (B) caspase-3 enzyme activation in NRK-52E cells following exposure of NiO@rGO nanohybrids. Arrowheads indicated that cells with fragmented nuclei/chromatin. *p < 0.05 vs. control.

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Detection of NiO@rGO nanohybrids induced apoptosis in NRK-52E cells through ao/etbr staining

Fluorescence dual staining of AO/EtBr probes can be utilized to detect apoptosis-associated alterations in cell membranes throughout the apoptosis process.68 AO entered normal cells with uninterrupted membranes and fluoresced green upon binding with DNA. However, EtBr penetrated the cells with disrupted membranes of apoptotic cells and produced orange-red fluorescence upon binding with fragmented DNA.69 According to cytological changes, green-stained cells are viable cells and have a perfectly arranged nucleus, whereas the cells that have undergone apoptosis have orange-to-red tinted nuclei with disintegrated or compacted chromatin.70 In this work, the apoptotic-inducing efficiency of the NiO@rGO nanohybrids in NRK-52E cells was experimented with by dosing the cells for 48 h with various levels of nanohybrids (1, 5, and 25 µg/mL) using AO/EtBr dual labelling. Results pointed out that the quantity of apoptotic cells (orange-red fluorescence) in the exposed group was greater than the controls, and the level of apoptosis augmented with increasing the dosages of NiO@rGO nanohybrids (Fig. 9). Our outcomes further advocate that NiO@rGO nanohybrids incite cell death in NRK-52E cells through the apoptosis pathway. A recently published work from our group also showed that CuO-decorated RGO nanocomposites induced apoptotic bodies in NRK-52E cells, as it was measured through AO/EtBr dual staining.30 Another recent work from Anushya and co-workers demonstrated that rGO-Bi2O3 nanocomposites provoke apoptosis in human lung cancer cells (A549 & H460).62.

Fig. 9
figure 9

Acridine orange and ethidium bromide (AO/EtBr) dual staining. Cells were observed to be viable (bright green), early apoptosis (light green fluorescence), and late apoptosis (orange to red fluorescence).

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NiO@rGO nanohybrids instigated cell cycle arrest in NRK-52E cells

Induction of cell cycle arrest at a particular checkpoint leading to apoptosis is a frequent process of cytotoxicity persuaded by nanoscale materials.71 The SubG1, G1, S, and G2/M are four main stages of the cell cycle.72 Cells with irreversible DNA damage are destined for apoptosis and accumulated sub-G1 phase. Earlier studies have demonstrated that metal oxide-based nanohybrids disturb the normal cell division process due to their ability to disrupt the regular progression of the cell cycle.73 In this study, flow cytometry was utilized to explore the consequences of NiO@rGO nanohybrids in cell cycle progression in NRK-52E cells. NiO@rGO nanohybrid-treated cells resulted in a considerably higher cell number in the sub-G1 phase than controls. Figures 10A-D represent the flow cytometry graph, whereas Figure 10E represents the quantitative inspection of different stages of the cell cycle in the control and exposed cells. As we can see in Figure 10E, after 48 h of exposure to NiO@rGO nanohybrids at dosages of 1, 5, and 25 µg/mL, the proportion of the sub-G1 population increased by 2.1, 3.2, and 8.3%, respectively, compared to the control (0.8%). The G1 stage of cell cycle was also dysregulated in NiO@rGO nanohybrids-treated NRK-52E cells. It is known that during the G1 phase, cells grow and DNA synthesis occurs. These results suggest NiO@rGO nanohybrids induced apoptosis in NRK-52E cells through dysregulation of cell cycle phases. These results are aligned with previous reports, in which researchers observed that metal oxide-based nanocomposites induce cell cycle arrest in different types of mammalian cell lines.30,62,65,68 Altogether, the effect of NiO@rGO nanohybrids on chromosomal condensation, caspase-3, and cell cycle arrest suggest that these parameters might be outstanding biomarkers to examine the genotoxicity of nanocomposites.

Fig. 10
figure 10

Cell cycle study of NRK-52E cells after treatment of NiO@rGO nanohybrids for 48 h. (A–D) Representative flow cytometry graphs demonstrating different phases of cell cycle. (E) Quantitative analysis of various stages of the cell cycle. *p < 0.05 vs. control.

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NiO@rGO nanohybrids induced cytotoxicity in A549 cells and HUVECs

Studies suggested that it is requisite to explore the effect of nanostructures on different cell types.74,75 Hence, to elude specific cell type reaction, we additionally explored the cytotoxic effects of NiO@rGO nanohybrids in HUVECs & A549 cells. These two cells have been commonly applied in nanomaterial cytotoxicity studies.76,77 The HUVECs and A549 were dosed to various levels (1–200 µg/mL) of NiO@rGO nanohybrids for 48 h. Figures 11A and B showed that NiO@rGO nanohybrids significantly induced cytotoxic effects in both HUVECs and A549 cell lines in a dose-reliant fashion. It was further noticed that NiO@rGO nanohybrids elevate ROS levels in both cell types (Figs. 11C and D). These results clearly indicated that NiO@rGO nanohybrids can generate cytotoxic effects in different types of mammalian cells.

Fig. 11
figure 11

Cytotoxic response of NiO@rGO nanohybrids in A549 cells (A) and HUVECs (B) exposed for 48 h. NiO@rGO nanohybrids induced ROS generation in A549 cells (C) and HUVECs (D) exposed for 48 h. Each value denotes the mean ± SD of three individual tests. *p < 0.05 vs. control.

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Plausible toxicity mechanisms of NiO@rGO nanohybrids

ROS-induced oxidative stress has been proposed as a possible mechanism of toxicity caused by nanoscale structures. Oxidative stress is defined as an imbalance between generated ROS and antioxidant defence capabilities of cells and tissues. Numerous studies have shown that ROS-induced oxidative stress is linked to a variety of diseases, including cancer, dementia, stroke, and cancer. ROS, such as superoxide anion, hydroxyl radical, and hydrogen peroxide, serve as signalling molecules for the onset and execution of apoptosis. Our findings suggest that NiO@rGO nanohybrid-induced cytotoxicity is due to its ability to create intracellular ROS and antioxidant GSH depletion, which leads to chromosomal condensation, cell cycle arrest, and apoptosis as a result of oxidative stress. Figure 12 depicts the plausible toxicity mechanism of NiO@rGO nanohybrids.

Fig. 12
figure 12

Plausible toxicity mechanism of NiO@rGO nanohybrids.

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Conclusion

Here, the cytotoxicity of NiO@rGO nanohybrids was explored in NRK-52E cells through oxidative stress, cell cycle arrest, and apoptosis routes. The NiO@rGO nanohybrids were produced by a simple hydrothermal protocol. The XRD, TEM, SEM, EDS, and DLS characterization data confirm the synthesis of high-grade NiO@rGO nanohybrids without impurities. MTT, trypan blue, and cell morphology data presented that NiO@rGO nanohybrids induced a dosage- and time-reliant cytotoxicity in NRK-52E cells. The NiO@rGO nanohybrids were also recognized to instigate oxidative damage, explicable by ROS elevation & GSH diminution. The NiO@rGO nanohybrids further induced apoptosis in NRK-52E cells, supported by chromosomal condensation, caspase-3 enzyme activation, generation of apoptotic bodies, and cell cycle arrest. Additionally, NiO@rGO nanohybrids induce significant cytotoxicity and ROS elevation in HUVECs & A549 cells, which suggests that the toxic potential of NiO@rGO nanohybrids is not cell-type-specific. Current research underlines the significance of potential toxicity of NiO@rGO nanohybrids and the critical need for comprehensive study in different cell types and suitable animal models. Understanding the plausible mechanisms of toxicity could aid in the development of safer nanocomposites for energy, environmental, and biomedical applications.